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Fluorescent probe and kit for abasic endonuclease 1 as well as application

A base nucleic acid and fluorescence detection technology, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high recovery rate, interference probe response, insufficient sensitivity, etc. High sensitivity and good stability

Active Publication Date: 2019-03-08
JIANGSU JITRI MOLECULAR ENG INST CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the method is not sensitive enough for serum samples with low APE1 content
When used for the determination of APE1 content in cell lysates, the recovery rate of the method is high, indicating that there are active substances in the sample that interfere with the response of the probe to APE1

Method used

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  • Fluorescent probe and kit for abasic endonuclease 1 as well as application
  • Fluorescent probe and kit for abasic endonuclease 1 as well as application
  • Fluorescent probe and kit for abasic endonuclease 1 as well as application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1

[0052] In the research results reported by APE1, the most suitable substrate for this enzyme is a DNA duplex containing an abasic site. With hydrolysis ability. This was also confirmed in our experiments. The DNA / RNA hybrid strand probes formed by hybridizing the D-1 strand and the RNA-probe strand in Table 1-1 did not respond to the increase of APE1 fluorescence. However, in our other experiments, we accidentally found that when the 5' direction of the abasic site in the DNA chain is a G base, the bacterial-derived similar enzyme EndoIV of APE1 can directly hydrolyze the DNA single strand, and when the abasic site When the 3' direction of the site has a weak binding effect on the complementary strand, it is beneficial for APE1 to attack the abasic site. Based on these reaction characteristics, we conceived an APE1 probe, which is a DNA / RNA hybrid probe, in which there is an abasic site on the DNA chain, and the 5' side of the abasic site is a G base , ...

Embodiment 2

[0065] Example 2

[0066]In this example, the reaction buffer of APE1 was optimized first. In the method reported before using DNA duplexes containing abasic sites as probes, the APE1 reaction buffer used was NEB buffer 1.1 (10mM Bis-Trispropane-HCl, 10mM MgCl 2 , 100 μg / mL bovine serum albumin) and then add 3.5 mg / mL human serum albumin. In this example, NEB buffer 4 (50mM KAc, 20mM Tris-Ac, 10mM Mg(Ac) 2 , 1mM DTT), on this basis, 0.04% (v / v) Triton X-100 was added to obtain a new reaction buffer.

[0067] The specific steps for detecting different concentrations of APE1 are as follows:

[0068] In the above-mentioned APE1 reaction buffer solution, 200nM APE1 probe and different concentrations of APE1 solutions were added respectively, and the solution was immediately detected in real-time fluorescence at 37° C. in a real-time fluorescent PCR instrument. According to the change curve of fluorescence intensity with time, the fluorescence rising rate is obtained, which is...

Embodiment 3

[0072] Example 3

[0073] We added different concentrations of biotin antibody to the reaction solution, and found that it could speed up the reaction rate between APE1 and the probe, further improving the detection sensitivity. The specific experimental steps are as follows:

[0074] In the reaction buffer solution optimized in Example 2, add the APE1 probe and biotin antibody solutions of different concentrations, mix well, add the APE1 standard solution, and immediately perform real-time fluorescence detection on the solution in a real-time fluorescent PCR instrument at 37°C . According to the change curve of fluorescence intensity with time, the fluorescence rising rate is obtained, which is the hydrolysis efficiency of APE1 to APE1 probe at the concentration of the biotin antibody solution.

[0075] The above reactions were all carried out in a 50 μL reaction system, the APE1 probe concentration was 200 nM, the biotin antibody solution concentrations were 0, 1, 2, 4, a...

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Abstract

The invention discloses a fluorescent probe and a kit for abasic endonuclease 1 as well as application thereof. The probe is a DNA / RNA hybrid probe, wherein a DNA chain is provided with an abasic site; the 5' side of the abasic site is G base; two mismatch bases continuously exist at the 3' side to form an outgrowth region; a fluorophore group and a quenching group are respectively marked on DNA chains at two sides of the outgrowth region. APE1 can recognize and cut a phosphoester bond in the 5' direction of the abasic site on the DNA chain and divide the DNA chain into two parts, so that thefluorophore group transmits a fluorescence signal and direct detection of the APE1 content in a complex biological sample environment is realized. Compared with an existing fluorescence analysis method, the fluorescent probe disclosed by the invention has the advantages that a probe structure and a reaction solution formula are greatly improved, the sensitivity is higher, and detection results aremore accurate and more reliable; in addition, a reagent is lower in cost and better in stability.

Description

technical field [0001] The present invention relates to the field of analysis and detection of apurinic / apyrimidinic endonuclease 1 (APE1), more specifically, to a detection kit based on an oligonucleotide fluorescent probe, and using the kit to detect biological samples Methods for the APE1 content in . Background technique [0002] Human apurinic / apyrimidinic endonuclease 1 (APE1 for short), also known as redox effector factor-1 (Ref-1 for short), is an important DNA repair proteins and gene regulatory proteins. On the one hand, it acts on apurinic / apyrimidinic sites (apurinic / apyrimidinic sites, referred to as abasic sites) in the DNA double strand, and is the most important abasic nucleic acid repair enzyme in the human body. pathway (base excision repair, BER) plays an important role; on the other hand, APE1 can also regulate the DNA binding activity of various transcription factors through its redox function. [0003] Studies have shown that APE1 is abnormally expre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2563/107
Inventor 赵美萍王嘉禹卢鹏李梦圆
Owner JIANGSU JITRI MOLECULAR ENG INST CO LTD
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